Mass spectrometric methods for the analysis of protein structures are being improved, applied and tested in independent and collaborative research projects in proteomics. Projects in separation methods and data processing are in progress. In the past year, progress has been made on implementation of a computational environment for mass spectrometric proteomic data processing;isotope labeled fusion proteins as protein standards;quantification of identified and unidentified components in sets of complex liquid chromatography-high resolution mass spectrometry data;understanding post translational modification in bacterial ribosomes;and multi-institutional collaborative studies on standards in proteomics. The processing and mining of mass spectrometric proteomic data requires a system allowing investigators desktop computer access to terabytes of data;the ability to repeatedly probe their data with emerging software tools;and the ability to transfer this data to public archival repositories. A versatile system has been implemented in LNT and will be shared with other proteomics groups in NIH. We sought to determine if biosynthetic concatenated labeled peptides (concatemers) are equivalent to whole labeled proteins as internal standards for isotope dilution mass spectrometry using selected reaction monitoring on a triple quadrupole mass analyzer. Mass spectrometry provides a platform for these measurements by using multiple reaction monitoring to follow specific transitions of peptides as they fragment;however, internal standards are only accurate if they faithfully mimic proteolytic properties of full-length proteins. We selected signature peptides plus 12 amino acids (6 amino- and 6 carboxy-terminal) through mass spectrometric screening as well as the public databases and literature. Synthetic genes for the extended selected sequences are fused with affinity tags and expressed by cloning a synthetic gene into an expression vector and labeled using 13C and 15N arginine and lysine amino acids. A human serum albumin (HSA) concatemer was tested because native HSA is readily available as well as 15N-labeled full-length HSA as a laboratory standard. HSA concatemer concentration was measured with respect to a chemically synthesized strep tag 10-mer peptide using a standard curve. Time, temperature, and enzyme studies were optimized. Three of the five peptides in the HSA concatemer accurately mimic the tryptic properties of native HSA. We demonstrated that concatenated HSA peptides can be used as internal standards for the quantification of HSA in urine samples. Traditional immunoturbidimetric data provided comparable results. For the immunoturbidimetric assays, 50 &#956;L of urine are consumed;for the selected reaction monitoring mass spectrometry method, less than 5 &#956;L are consumed. New algorithms that serve as the basis for the bioinformatics tools used in the analysis of liquid chromatographic - tandem high resolution mass spectrometry data resulted in several open source software tools becoming viable alternatives to proprietary software. Unbiased protein analysis has been required to assess differences between control and treatment groups in several of our projects. The determination of relative expression levels of peptides in large data sets with biological replicates has remained challenging. The size and complexity of the data generated by LNT has consistently pushed the envelope of all the existing software. Replicate LC/MS/MS runs at three concentrations of the Universal Proteomics Standard (Sigma UPS1) were acquired in profile mode at a resolution of 60,000 on an LTQ-Orbitrap. Resultant spectra were analyzed using MZmine, XCMS, Mass++, and Progenesis LC-MS in both profile and centroid mode. Additional replicate LC/MS/MS runs at a set concentration of UPS1 at each of four increasing background concentrations of Human Serum Albumin were also acquired. Data was analyzed with respect to performance with large dynamic range, and ease/accuracy of LC/MS/MS alignment. Ideal-Q, a software tool which integrates a database search requirement with AMT based assignments was also used for the analysis. The integration of features results in overall stable ratios, although the statistical spread of the data is extremely variable. The use of Accurate Mass Tags to share database assignments across multiple replicates enables a more complete description of the data. Studies on the functional characterization of the post-translational modification (PTM) beta-methylthio-aspartic acid of the Escherichia coli at position 88 in protein S12 have been completed. There is a correlation between the presence of this PTM and the transcription of anaerobic genes belonging to the FNR regulon. Mass spectrometry analysis and affinity pull-down assays were used to identify two proteins RimO and YcaO that are specifically linked to interacting with and modifying S12. Gene knockouts for both proteins revealed a dramatic decrease in the modified form of S12 (the RimO knockout resulted in a complete absence of modified S12) and an overlapping decrease in transcription of genes belonging to the FNR regulon (determined by DNA expression microarray). Further investigation revealed that an absence of the PTM results in a substantial decrease in the abundance of the transcriptional factor FNR required for these genes to be expressed. It therefore seems likely that the S12 PTM affects the mRNA specific translation of FNR although the molecular details are unknown.